Studies are described that are designed to identify conditions of mixed organic-aqueous solvents and subzero temperatures under which true reaction intermediates of liver alcohol dehydrogenase can be stabilized and structurally characterized. Synthesis of the chromophoric alcohol 4-(2-imidazolyl)-azobenzylalcohol is described for use as a direct kinetic probe of alcohol oxidation in cryoenzymologic studies. This synthetic substrate should exhibit moderately intense absorption in the near ultraviolet (350-400 nm) region that is non-overlapping with that of the coenyzme NAD+. Once cryosolvent conditions are identified on the basis of cryokinetic experiments for stabilization of the catalytically competent enzyme-NAD+ alcohol complex of the native enzyme, they shall be applied to the active site specific Co++ reconstituted enzyme for structural characterization of the coordination environment of the active site metal ion by electron paramagnetic resonance methods. The conditions for stabilizing reaction intermediates will be applied also to determine the configuration of the substrate in the catalytically competent enzyme-NAD+ alcohol complex. For this purpose, the synthesis of the spin-label nitroxide substrate 3-(2,2,5,5-tetra-methylpyrrolinyl-1-oxyl)-propen-1-o1 is described for use as a spectroscopic probe. With use of the Co++ reconstituted enzyme, the metal ion-to-nitroxide distance in the enzyme-NAD+ alcohol substrate complex stabilized at subzero temperatures can be determined on the basis of the dipolar interactions that quench the EPR signal amplitude of the nitroxide moiety. This quantitative structural parameter can be then employed to search out stereochemically compatible configurations of the substrate in the active site by molecular graphics techniques. Direct determination of the structural environment of the active site metal ion in catalytically competent reaction intermediates will enable assignment of the structural basis of alcohol oxidation catalyzed by liver alcohol dehydrogenase.